Abnormal proteins and many short-lived regulators are targeted for degradation by a number of ATP-dependent protease machines. These machines all appear as large oligomeric cylindrical Complexes in electron micrographs. The three archetypes are the 26S proteasome, Clp and Lon. This proposal focuses on structural and mechanistic studies of two bacterial protease machines (Clp and HsIUV) that represent two of the three archetypes. The Clp protease is composed of a proteolytic core, ClpP and an ATPase component, ClpA, ClpC or ClpX. HsIUV is a hybrid comprising a proteolytic component, HsIV (ClpQ) that is homologous to the beta-type subunits of the 265 proteasome, and a Clp- like ATPase component, HsIU (ClpY). The Clp ATPase subunits exhibit chaperone activity both in vitro and in vivo and are capable of facilitating both the folding/activation and the degradation of proteins. As such, these ATPases may be an important decision point in these cellular pathways. In addition to the 205 protease, HsIV, is one of the defining members of a new class of hydrolytic enzymes, termed the Ntn-hydrolases (N-terminal nucleophile), in which the terminal threonine residue acts as the nucleophile. The Clp and HsIUV systems have major advantages over Lon and the 26S proteasome as a model system. In Lon, both the proteolytic and ATPase components reside with in a single polypeptide chain making substrate recognition and proteolysis an extremely tightly coupled process. In our model systems, the proteolytic and presenting ATPase components exist as stable homo-oligomers that associate transiently in the presence of ATP to degrade proteins. This allows us to separate, express and manipulate each component and determine the structures individually. In the 26S proteasome, each component is hetero-oligomeric and thus phasing information must be gained using standard x-ray techniques. In our systems, the components are homo-oligomeric and crude, low resolution, models can be used to determine initial phases and extended to atomic resolution by exploiting non-crystallographic symmetry averaging techniques. We have developed this technique and demonstrated its viability in the published structure of ClpP. We will use this approach again in the determination of HsIV(ClpQ) for which we have data to 3.0 Angstroms resolution. By symmetry averaging we can solve the structure and any deviations of the molecules from noncrystallographic symmetry can be seen in the electron density maps at high resolution (>2.5 Angstroms). Indeed, further refinement of the ClpP structure identified an asymmetry that may account for the preferential binding of ClpA to one face of the ClpP cylinder. On the basis of this structure, we have proposed a conserved mechanism for energy-dependent proteolysis. This proposal focuses on determining the structures of individual components of the proteolytic machinery (both proteolytic and ATPase) and testing our hypothesis.